Integrative Biology

REVIEW ARTICLE

Synthetic biology approaches in

immunotherapy, genetic network engineering, Downloaded from https://academic.oup.com/ib/article-abstract/8/4/504/5163774 by Columbia University user on 27 March 2020 Cite this: Integr. Biol., 2016, 8, 504 and genome editing

Deboki Chakravarti,* Jang Hwan Cho,† Benjamin H. Weinberg,† Nicole M. Wong† and Wilson W. Wong*

Investigations into cells and their contents have provided evolving insight into the emergence of complex biological behaviors. Capitalizing on this knowledge, synthetic biology seeks to manipulate the cellular machinery towards novel purposes, extending discoveries from basic science to new applications. While these developments have demonstrated the potential of building with biological parts, the complexity of cells can pose numerous challenges. In this review, we will highlight the broad Received 24th December 2015, and vital role that the synthetic biology approach has played in applying fundamental biological Accepted 31st March 2016 discoveries in receptors, genetic circuits, and genome-editing systems towards translation in the fields DOI: 10.1039/c5ib00325c of immunotherapy, biosensors, disease models and gene therapy. These examples are evidence of the strength of synthetic approaches, while also illustrating considerations that must be addressed when www.rsc.org/ibiology developing systems around living cells.

Insight, innovation, integration Investigations into cells and their contents have provided evolving insight into the emergence of complex behaviors. Capitalizing on this knowledge, synthetic biology seeks to manipulate the cellular machinery towards novel purposes, extending basic discoveries from basic science to new applications. While these developments have demonstrated the potential of building with biological parts, the complexity of cells can pose numerous challenges. We will highlight the role that the synthetic biology approach has played in applying fundamental biological discoveries in receptors, genetic circuits, and genome-editing systems towards translation in the fields of immunotherapy, biosensors, disease models and gene therapy. These examples illustrate the strength of synthetic approaches, as well as considerations that must be addressed when developing systems around living cells.

Introduction areas: sensors, genetic circuits, and genome controls. Sensors allow engineered cells to detect and respond to a variety of Building basic tools with synthetic biology extracellular and intracellular signals, such as cancer antigens,1,2 Cells regularly perform immense tasks, processing signals light,3 and small molecules.4,5 Since the seminal works of the from many sources to gauge and execute a proper response. synthetic toggle switch6 and oscillator,7 many synthetic genetic Over several decades, our knowledge of the components and circuits have been built to program both prokaryotes and connections that underlie these calculations has expanded in eukaryotes with synthetic, complex decision-making systems many organisms, providing the foundation to engineer novel such as logic gates,8 classifiers,9–11 edge detectors,12 counters,13 behaviors into cells. One of the major aims of synthetic biology controllers,14 and finite state machines.15 These and parallel approaches is to translate these discoveries into works have been complemented by the advancement of genome well-characterized and reproducible components of molecular editing tools that have made rapid genome modification feasible engineering. in many species.16–19 The results from work in all three of these While many such engineering parts have been developed areas have produced tools that can be applied towards both the with synthetic biology, this review will focus on three major understanding and engineering of biology.

Interrogating biology using synthetic tools Department of Biomedical Engineering, and Biological Design Center, Boston University, Boston, Ma, USA. E-mail: [email protected] The construction of synthetic molecules and circuits has † Equal contribution. enabled scientists to explore the complex networks underlying

504 | Integr. Biol., 2016, 8, 504--517 This journal is © The Royal Society of Chemistry 2016 Review Article Integrative Biology cellular behavior. These studies often fall into either a ‘‘reverse’’ The principles of synthetic biology have taken shape in these or ‘‘forward’’ engineering approach.20 In the ‘‘reverse’’ engi- areas through examinations on the modularity of biology neering strategy, networks in a cell are perturbed at different (chimeric antigen receptors), the use of iterative, computationally- nodes,21 and the resulting changes in behavior provides insights driven design (synthetic oscillators), and the ability to develop tools into the role of different genes and proteins. The complementary for widespread use (genome editing). These characteristics are not ‘‘forward’’ engineering approach enables scientists to hypothesize mutually exclusive, and themes that describe one of these areas and test different design criteria by programming artificial systems of research may be visible in the others. These works also reflect into a cell.22 For instance, artificial feedback loops were generated the fundamental requirement and challenge of synthetic biology: to study the topology involved in controlling the Bacillus subtilus implementation of an engineered system with an environment that 23 competence network dynamics, and synthetic ‘‘secrete and is both complex and not fully understood. The challenges that have Downloaded from https://academic.oup.com/ib/article-abstract/8/4/504/5163774 by Columbia University user on 27 March 2020 sense’’ circuits were developed to investigate the onset of social arisen in these three areas have highlighted gaps in our knowledge or asocial behaviors in yeast.24 and provided further insight into the relevant parameters for these technologies. These new approaches to biology are accompanied Toward commercial applications of synthetic biology by serious questions regarding their safety and practicality. In In addition to elucidating fundamental design principles, syn- assessing the scientific, economic, and ethical considerations that thetic biology has sought to apply the principles of engineering are part of the synthetic biology process, we can gain a deeper cellular behavior towards major challenges in areas such as health understanding of how to build genetic technologies that are and the environment. In some cases, synthetic biology has even applicable to real world challenges. been able to play a major role in commercial product development. Yeast and bacteria are attractive factories to manufacture chemicals that can be otherwise expensive to produce. These organisms Chimeric antigen receptors grow quickly, can be scaled towards large-scale production, and—most importantly for synthetic biology—are relatively Chimeric receptors to understand T cell signaling easy to engineer.25,26 Yeast have been modified through genetic Our adaptive immune system regularly performs complex calcu- engineering to produce the immediate precursor to the potent lations with tremendous consequences for our health. T cells are antimalarial drug artemisinin, providing more stability to the vital to this system, driving the detection, elimination, and production of a drug that was previously reliant on extraction memory of pathogens through processes that are largely directed from the wormwood plant Artemisia annua and thus subject to by the T cell receptor (TCR). The TCR assesses other cells for plant conditions.27,28 The pharmaceutical company Sanofi has signs of pathogen invasion by analyzing epitopes—short peptide licensed the engineered yeast strain to produce more than 39 sequences excised from proteins in the antigen-presenting tonnes of the drug, which can be used for more than 40 million cell (APC)—positioned on the cell surface by the major histo- treatments. Similar strategies to program synthetic pathways in compatibility (MHC) complex. Activation through the TCR is microbes have been developed to produce opioids in yeast29 MHC-restricted, meaning that the receptor must bind to an and biofuels in microorganisms.30–33 MHC-peptide complex to trigger the T cell. In addition to metabolite production, synthetic approaches In the 1980s, the first chimeric receptor to trigger T cell have been critical in the development of cancer therapeutics activation was developed and expressed39 (Fig. 1A). Connecting and disease diagnostics. The engineering of T cells using the variable regions of an antibody to the variable chains of the synthetic cancer-targeting receptors has been hailed as a break- TCR, these chimeric receptors were able to detect antigens and through in cancer therapy due to its unprecedented efficacy activate the T cell upon target-binding (Fig. 1B). This response against leukemia.34 Genetic Boolean logic circuits have been was dictated by the binding of the variable antibody region to expressed in to detect glycosuria in urine from the target antigen, indicating that this chimeric receptor- diabetic patients,35 and synthetic circuits have been adopted in mediated T cell activation was not MHC-restricted. The clinical a cell-free and paper-based format for the rapid and low cost potential of this receptor to treat cancer was evident and noted detection of various chemicals and Ebola viral RNA.36 in the paper. However, long before the appearance of promising clinical data, chimeric receptors became a powerful research Learning from the synthetic biology process tool to study the proteins involved in T cell signaling. Synthetic biology is a vibrant field, and many reviews have The TCR contains variable a and b chains in complex provided surveys of the fundamentals and applications of the with several invariant proteins. One of the early goals in TCR field.20,25,37,38 The goal of this review will instead be to high- research was to decipher the roles of these invariant proteins light synthetic biology as a framework to bridge fundamental activating the T cell. In the 1980s, the variable chains of the studies of biology with applications. We will illustrate this TCR were known to complex with CD3 chains,40–44 and further viewpoint by summarizing the developments of the chimeric studies illustrated that the CD3z chain in particular was antigen receptor, synthetic gene circuits, and genome editing coupled to molecules engaged in signal transduction.45,46 However, tools, which each reflect developments in the three major while the association between the TCR a and b chains with the categories of synthetic systems described earlier (sensors, circuits, CD3z chains was clear, the specific role of CD3z in T cell activation and chassis). was not well understood.

This journal is © The Royal Society of Chemistry 2016 Integr. Biol., 2016, 8, 504--517 | 505 Integrative Biology Review Article

original chimeric receptor. In place of antibody regions, the extracellular domain of these chimeric receptors contained regionsofproteinssuchasCD1647 or CD848,49 that have known targets for antibody-binding. Meanwhile, the intracellular domain of the receptor was comprised of the CD3z chain (or in some studies, fragments of the CD3z chain)(Fig.1B).Inisolatingthis chain from the TCR complex, these chimeric receptors enabled studies on the involvement of CD3z in activation upon binding of an antibody to the extracellular domain.

These chimeric receptors provided significant insights into Downloaded from https://academic.oup.com/ib/article-abstract/8/4/504/5163774 by Columbia University user on 27 March 2020 TCR design, demonstrating that the fusion of the intracellular region of the CD3z chain to the extracellular binding domain was sufficient to trigger activation.49 Similar experiments using fragments of the CD3z in place of the full domain identified the 18 residue active site of the chain.47,48 These efforts reflect the ability to use synthetic molecules to identify key characteristics of relevant molecules. The ability to turn these early chimeric receptors against a therapeutic target was apparent when a chimeric receptor specific for HIV envelope protein was able to direct cytolytic activity against target cells.50

Therapeutic applications of synthetic receptors The knowledge obtained from chimeric receptor studies fed back into the development of chimeric antigen receptors (CARs) for cancer therapy. The performance of CD3z in the chimeric receptors for TCRs supported its potential for use as part of the therapeutic design,51,52 and the first generation of CARs were a fusion of the CD3z domain to the single chain variable fragment (scFv) from an antibody (Fig. 1B). However, initial clinical results indicated that the first-generation CARs were unable to persist in patients long enough to trigger a therapeutic response53,54 This initial CAR design reflected a focus on the antigen- dependent aspect of T cell activation. However, T cell activation requires several signals. The binding of the receptor to a target antigen is one signal, but this step is supplemented by a co-stimulatory trigger that is generated by the interactions of surface molecules expressed on both the T cell and the APC. Previous studies demonstrated that chimeric receptors could be designed to trigger co-stimulation. In 1996, a synthetic

Fig. 1 Synthetic biology approaches for engineered receptors and activation receptor was designed to make CD28—a costimulatory glyco- controls. (A) Timeline of engineered receptors. (B) Chimeric receptors were protein expressed on the surface of T cells—antigen-specific created to help study T cell activation. Further development on their design has through the construction of a scFv-CD28 molecule.55 This led to their use in therapeutic applications, for example the use of chimeric chimeric receptor elicited a co-stimulatory response with the antigen receptors. (C) Knowledge of how T cells activate through receptors endogenous TCR complex that was comparable to wild type CD28. and downstream pathways has allowed for the development of various receptor domains and genetic switches to control T cell activity. Examples of Furthermore, when the chimeric receptor was co-transfected with this are the pause switch using bacterial effector proteins YopH and OspF; CAR another scFv-CD3z receptor, the signals from these two chimeric using CTLA-4 and PD-1 inhibitory domains; split CAR to target multiple receptors were able to mediate a T cell response, indicating that antigens; and an ON-switch using small molecule activation. (D) Chimeric the CD28 and CD3z domains could be engineered together antigen receptors are a modular system for engineering T cell control. to drive optimal T cell response. The intracellular domain of Individual components can be modified to change the overall behavior of z the receptor. The main modules of the CAR consist of a targeting domain, CD28 was integrated with CD3 in 1998 to produce a multi- 56 signaling domain, and downstream controls. domain chimeric receptor. Today, this CAR is considered a ‘‘Second Generation’’ CAR due to its integration of a single co-stimulatory domain with the activating CD3z domain (Fig. 1B). The chimeric receptors developed to study T cell signaling The repertoire of these second generation CARs has grown to domains were constructed with different domains from the include other co-stimulatory domains, including 4-1BB57 and OX40.58

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This structure has been further expanded in ‘‘Third Generation receptors into multiple components. For example, while the CARs’’ to include multiple co-stimulatory domains in one scFv-CD28 and scFv-CD3z experiments originally demonstrated receptor57–59 (Fig. 1B). the co-stimulatory role of CD28, these results also indicated that multiple chimeric receptors containing different signaling Synthetic approaches to advancing therapy domains can synergistically drive activation. By choosing scFv’s These advancements reflect a modular approach to receptor for this split receptor design that target different antigens, construction that has brought chimeric antigen receptors full activation of the T cell requires both the scFv-CD3z CAR (CARs) to the forefront of cancer research. T cells are uniquely and the scFv-CD28 chimeric co-stimulatory receptor to detect equipped with the ability to kill other cells, and CARs can their respective antigens68 (Fig.1C).Thiscombinatorialactivation

effectively ‘‘teach’’ a patient’s T cells to detect and eliminate system can thus increase T cell specificity towards a tumor. Downloaded from https://academic.oup.com/ib/article-abstract/8/4/504/5163774 by Columbia University user on 27 March 2020 cancer cells. Despite the poor clinical performance of first- CARs can also be split into two halves to allow greater control generation CARs, the improvements made in second-generation over when the T cell will activate, as implemented in the CARs drove such strong responses against B cell malignancies ON-switch CAR. Instead of dividing the CAR into two separate thatseveralclinicaltrialshave elicited up to a 90% complete chimeric receptors, the ON-switch CAR splits the CAR into two response rate in patients.60–62 However, the onset of toxicities separate domains—antigen-binding and intracellular signaling including cytokine storms and fatal off-tumor responses exposes domains—that attach in the presence of a drug69 (Fig. 1C). Even the limitations of this therapy as well.61,63 if the antigen-binding domain binds to the target antigen, it Underlying these toxicities is a fundamental question of will not activate the T cell until the drug has been added and cell-based therapies in their current state: once the cells have the intracellular signaling domain has attached. Further efforts been transfused back into the patient, how can a desired to improve CAR-based therapy with synthetic biology are behavior be ensured? Current approaches to cytokine storms ongoing, and as researchers from both sides work to develop require the use of steroids to weaken the immune system and technologies that will make this treatment safe and viable for reduce the onset of cytokine release syndrome (CRS)-related many patients, the melding of these approaches may provide symptoms.60 However, compared to the relative simplicity of a greater understanding of both the immune system and the chemical drugs, cells require more powerful safety strategies to design of receptors. match their own complexity and provide the most effective treatment for an individual patient. The synthetic approach underlying the development of CARs Genetic circuits has played a prominent role in the development of more powerful controls over adoptive T cell therapy, creating an Applications of genetic circuits towards medicine attractive arena for synthetic biologists and immunologists to Gene networks are vital for the regulation of cellular behavior. collaborate. For example, the bacterial virulence proteins OspF The topology of these networks can have important implications (outer Shigella protein F) and YopH (Yersinia outer membrane) for processes that take place when a cell is establishing its have been introduced into T cells to create a drug-inducible identity, all the way through its death. One of the natural goals ‘‘pause switch’’ due to their ability to inhibit kinases involved in of synthetic biology—a field built out of genetic engineering—is TCR signaling64 (Fig. 1C). Synthetic approaches can addition- to apply the principles underlying the behavior of these circuits ally be implemented to control the growth or viability of T cells, towards the design of novel systems. Novel gene circuits have such as a ribozyme switch to regulate expression of interleukin turned organisms into pharmaceutical and biofuel factories, as (IL) 2 or IL 15,65 or the inducible kill switch iCaspase9, which described earlier, as well as playing a role in the advancement of has been implemented to treat graft-versus-host disease (GvHD) therapeutics. The drug-inducible ‘‘pause switch’’ described in in stem cell transplant patients.66 the discussion of CAR therapies illustrates the potential for The underlying promise of modularity that has made CARs genetic circuits to enable bedside control of cell-based therapies, successful has continued to drive the design of novel receptors as does the riboswitch designed to provide control over T cell with additional controls (Fig. 1D). By altering the signaling proliferation.65 Synthetic gene circuits have also formed the basis domains of the chimeric receptors, responses other than activation of multiple strategies to treat diabetes. In one approach, the can be programmed into the cell for a given target. For example, expression of hormone that mediates blood glucose homeostasis instead of utilizing signaling activation and co-stimulatory in type 2 diabetes is activated by a signaling cascade triggered by domains, chimeric receptors assembled with inhibitory domains blue light, which was able to provide optogenetic improvement of like CTLA-4 or PD-1 block T cell activation upon antigen-binding67 blood-glucose homeostasis in a type 2 diabetic mouse model.70 (Fig. 1C). These inhibitory CARs (iCARs) have the potential to Another strategy that has been tested in mice for mediation increase specificity in immunotherapy by blocking the T cell from of blood glucose levels is through circuit that produces insulin attacking certain tissue without completely shutting down the in response to radio-waves.71 Synthetic circuits have also been treatment. considered as a tool to treat cancer, such as the microRNA-based While we have largely discussed modularity in the context of cancer cell classifier that uses microRNA expression to identify adding domains together in a direct fusion, this property can cancer cells and trigger apoptosis.72 And while these circuits have also take effect when considering the splitting of the chimeric all been implemented within a cell, they may also have great

This journal is © The Royal Society of Chemistry 2016 Integr. Biol., 2016, 8, 504--517 | 507 Integrative Biology Review Article power when applied in other mediums. For example, the ability to Consisting of three transcriptional (TetR, LacI, and program toehold switch mRNA sensors onto paper has opened lCI) arranged in a cyclic negative feedback loop, the repressilator up the potential to quickly produce sequence-based sensors at a was the first synthetic cellular oscillator7 (Fig.2AandB).With low cost.36 this system, 40% of cells showed oscillations of GFP in E. coli. However, performance of the repressilator was not optimal; noise Taking inspiration from designs in nature in gene expression resulted in large variability in the amplitude These systems all reflect an ability to construct genetic circuits andperiodofoscillations,andthe circuit was unable to produce towards an application. However, synthetic biology often involves oscillations at the stationary phase of cell growth. the iterative process of constructing and refining the design of To produce a more robust oscillator in E. coli, a positive

a system through many different systems and components. feedback loop was introduced in the general structure of the Downloaded from https://academic.oup.com/ib/article-abstract/8/4/504/5163774 by Columbia University user on 27 March 2020 This process often takes place within a lab, with the published activator/inhibitor relaxation oscillator92 (Fig. 2B). Containing work not reflecting the many designs that were attempted and both activator (NRI) and repressor (LacI) modules, the oscillation discarded on the route to the final product. The synthetic period was tunable by altering the nutrients in the medium. More oscillator—a foundational construct of the field7—enables a importantly, the system was robust to noise and able to function macroscopic view of improving on a design over time, as in cells growing at the stationary phase. The tunable relaxation multiple works have been published over several decades to oscillator added a second autonegative feedback loop to the improve the performance of the circuit and create various structure, driving oscillations in approximately 99% of the cells implementations, ultimately opening up its potential use for in the population93 (Fig. 2B). These cycles were also preserved application. after cell division and tunable, this time by varying temperature Oscillations are a common network in nature, causing the and inducer concentration. However, synchronization between appearance of a particular output to be cyclicle. Examples range the cells was limited. from chemical, such as the Belousov–Zharbotinsky reaction, In nature, the behavior between cells in a population can be to much larger systems, including the mathematically well- coordinated through quorum sensing. Relying on communica- characterized predator–prey ecosystem model.73 Within cells, tion to synchronize oscillation dynamics between the cells, this many signaling pathways contain components exhibiting oscil- mechanism provided inspiration for several synthetic designs latory behavior, such as the tumor suppressor p53,74,75 NF-kB,76 to reduce population variability. In a micro-chemostat system and cAMP,77 and the Cdk1–APC system controlling cell division.78,79 designed to maintain E. coli density, the LuxI/LuxR system from Periodic gene expression is also observed in budding yeast growing Vibrio fisheri enabled cells to communicate with each other under nutrient-limited conditions.80 Circadian rhythms in many via acyl-homoserine lactone (AHL) accumulation in the media, organisms are controlled by oscillating genes, such as the which triggered the expression of a kill gene in receiving KaiABC gene cluster in cyanobacteria,81 the per and tim gene cells.94,95 This AHL-mediated quorum sensing also provided in Drosophila,82,83 and the frq gene in Neurospora.84 Additionally, negative feedback in a predator–prey ecosystem analog in oscillations can drive synchronous behavior between organisms, bacteria. Utilizing LuxI/LuxR and LasI/LasR (derived from such as the rhythmic flashing of fireflies85 and the acetaldehyde- Pseudomonas aeruginosa), prey cells produced a signaling driven glycolytic oscillations in yeast.86 molecule that rescued predators96(Fig. 2C). Meanwhile, predator The prevalence of oscillators correlates to their significance cells produced a signaling molecule that killed prey cells. Quorum in biology. Plants rely on circadian dynamics to coordinate sensing has appeared in other designs as well due its ability to expression of certain genes to the light–dark cycle.87 In addition, synchronize the behavior of cells97,98 (Fig. 2C). the frequency or amplitude of oscillations can encode temporal Oscillator circuits have also been designed in E. coli information, as is observed in the transient signaling of by integrating metabolic and genetic parts (Fig. 2B). This calcium.88,89 While calcium ions are a common signaling ‘‘metabolator’’ contains transcriptional elements whose activities molecule, they are toxic at high, sustained concentrations. are regulated by outputs from the acetate pathway, producing Encoding information on the amplitude (amplitude modulation, oscillations that were observed in approximately 60% of the AM) or frequency (frequency modulation, FM) of calcium oscilla- cells and unaffected by cell division.99 However, this circuit was tions can potentially enable the cell to decipher information subject to variability due to stochasticity in gene expression. from the transient presence of the ion. For example, B cells rely Oscillators were also implemented in mammalian cells using on AM signaling to decide between several different pathways of the tetracycline- and pristinamycin-controlled activation.90 systems, though this system was also subject to variability100 (Fig. 2B). As synthetic biology makes advances in engineering Designing synthetic oscillators in cells new systems and organisms, the same spirit underlying this The primary design requirement to drive oscillations is the process that produced more robust oscillators may help to presence of a negative feedback loop with some delay.91 However, elucidate many other circuit designs. the inclusion of this motif does not guarantee optimal perfor- mance of the overall circuit, particularly in cells where factors Advancing oscillator design towards an application such as cell division and stochastic gene expression can affect the Due to their natural role in relaying environmental cues, network. oscillators hold promise as integral components in biosensors.

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Fig. 2 Topologies, population based and application of the oscillators. (A) Timeline of synthetic oscillators. (B) Topologies of different oscillators. (i) Repressilator. Three repressor genes were used to repress other . TetR represses lcI, and lcI represses LacI. LacI represses TetR. (ii) Activator/inhibitor relaxation oscillator. This design contains both activator (NRI) and inhibitor (LacI) modules. This design utilizes a positive autoregulatory circuit. (iii) Tunable relaxation oscillator. The second autonegative feedback was introduced to increases robustness of oscillator. (iv) Mammalian oscillator. Used both positive and delayed negative feedback. (v) Metabolator. This design utilize both transcriptional regulation and metabolic flux. (C) Population based oscillators. (top) Synthetic prey– predator system using bacterial quorum sensing signals. In prey cells, first QS molecule, 3OC6HSL (colored in yellow circle), is synthesized by LuxI and binds to transcriptional regulator LuxR to increase antidote gene expression (ccdA). In predator cells, second QS molecule, 3OC12HSL (colored in green circle), is synthesized by LasI and binds to LasR to activate kill gene (ccdB). (bottom) Synchronized oscillators. AHL QS molecule is synthesized by LuxI, which then binds to transcriptional regulator LuxR to regulate expression of LuxI and aiiA. aiiA gene degrades AHL and acts as negative regulator. (D) Arsenic sensing genetic biosensor. (i) Network diagram of sensing array. luxI synthesize QS molecule, AHL, that regulate gene expression of luxI, aiiA,andndh. ndh gene synthesizes enzyme that generates H2O2 which acts as second QS molecule that activates expression of luxI, aiiA,andndh. (ii) Network design of arsenic sensor. In thresholding (left), sensor reports the presence of arsenite above certain concentration. Here, luxR gene is removed from sensing array network, and it is now controlledbyarsenite-responsivepromoterthatisrepressedbyArsRinabsenceofarsenite.Inperiodmodulation(right),sensorisabletoreportthe concentration of arsenite through frequency of oscillation. Here, luxI is under arsenite responsive that affects oscillation period.

This journal is © The Royal Society of Chemistry 2016 Integr. Biol., 2016, 8, 504--517 | 509 Integrative Biology Review Article

The work described to implement and refine synthetic oscillators has made it possible to apply them towards this purpose. One example is the development of an arsenic sensor using E. coli,101 which relies on quorum sensing to synchronize the output of thousands of bacterial ‘‘biopixel’’ colonies (Fig. 2D). Two arsenic-sensing modules were designed with this capability: one that can report the presence of arsenic above a certain concentration, and another that is able to report the concentration of arsenic through the frequency of GFP reporter oscillations (Fig. 2D).

The designs of new oscillators that build upon previously Downloaded from https://academic.oup.com/ib/article-abstract/8/4/504/5163774 by Columbia University user on 27 March 2020 established systems are emblematic of the iterative process that underlies all engineering. The primary focus of these efforts over time was to increase the robustness of the circuit towards stochastic fluctuations and growth dynamics, as well as to reduce cell-to-cell variability. With each new design, the addition of new components and connections helped to improve the behavior of the overall circuit. The oscillator is thus an excellent case study in the exploration of new design rules over time to combat some of the challenges posed by biological noise.

Genome editing and control Bacteriophage and bacteria for genome editing technologies The interaction between invasive organisms and their attempted hosts can often turn combative. Our own bodies have evolved a complex machinery of defenses against pathogens, executing innate and adaptive commands to both detect and eliminate threats. Bacteria may not have the same capacity to develop this extensive immune system, but they also mount their own strategies against bacteriophage. For these organisms, the genome is the battlefield. Bacteriophage attempt to integrate their genetic material into the target cell, and the infected bacteria respond by cutting these sequences out. Both of these processes require enzymes that can target and cut specific DNA sequences, and investigations into the interactions between bacteriophage and bacteria have driven the development of several tools for genome editing and control (Fig. 3A and B). Bacteriophage-derived enzymes such as the lambda tyrosine integrase,102 Cre recombinase,103–105 PhiC31,106,107 and Bxb1108 have allowed for site-specific control over inversion, excision, and translocation of DNA (Fig. 3C). The ability to use these enzymes in vitro109 and in other organisms, including yeast and mammalian cells110,111 have made them useful tools in engineering downstream applications, such as Fig. 3 Genetic tools for genome engineering. (A) Timeline of recombinases disease models based on cell-specific expression of a particular and integrases. (B) Timeline of genome editing proteins. (C) Site-specific gene.112,113 recombinases are commonly used to delete or invert DNA sequences through two often used classes. (D) Site-specific nucleases, such as ZFN, While recombinases are powerful, their specificity is already TALEN, and CRISPR/Cas9 use different DNA binding elements to achieve programmed. Two systems for targeted genome editing have imprecise DNA deletions or insertions through non-homologous end joining been developed with the bacterial nuclease Fok1, which dimerizes or precise DNA addition through homology directed repair. (E) Catalytically to cleave DNA (Fig. 3D). One system utilizes transcription activator- dead Cas9 (dCas9) can be fused to transcription activation (AD) or repression like effectors (TALEs), a bacterial protein involved in infecting (RD) domains to enable activation or repression of genes, respectively. (F) Multiple guide RNAs can be used for multiplexed gene activation or different plants.114 By altering two residues in the TALE and then repression. Multiple guide RNAs can also be designed to target a promoter fusing the modified protein to the Fok1 nuclease catalytic to achieve higher levels of gene activation in a synergistic fashion. (G) Using domain, DNA cleavage can be targeted towards a desired a shorter gRNA target sequence (14nt) abolishes Cas9-mediated nuclease sequence with this transcription activator-like effector nuclease activity yet permits DNA-binding and activation of genes through fusion of a (TALEN). A similar strategy has been devised with zinc fingers, transcription activator.

510 | Integr. Biol., 2016, 8, 504--517 This journal is © The Royal Society of Chemistry 2016 Review Article Integrative Biology which contain a Cys2–His2 zinc finger DNA-binding motif, (such as the KRAB domain), and then guiding the dCas9 in place of TALEs. This design is advantageous because zinc towards a desired promoter (Fig. 3E).18,130,131 By expressing finger nucleases are small and can be easily engineered to several sgRNA targeted towards different promoters, this system target a desired sequence when connected to the Fok1 catalytic can activate or repress several promoters in a multiplexed domain.115 In addition to genome cutting, both TALEs and zinc fashion (Fig. 3F).132 The field of synthetic biology has a vested fingers have been engineered with other effector domains to interest in improving CRISPR in these various functions, and trigger targeted gene activation and repression.115–118 the exploration of new domains such as the SunTag (a peptide Recombinases and Fok1 are both derived from the bacterial array that can recruit antibodies fused to VP64)133 and VPR (a ‘‘innate’’ defenses. However, a broader picture of a bacterial fusion of VP64, p65, and Rta)134 have helped improve the gene

adaptive immune system has been illustrated following the activation capability of dCas9. The construction of split-Cas9s Downloaded from https://academic.oup.com/ib/article-abstract/8/4/504/5163774 by Columbia University user on 27 March 2020 observation of repeats within the bacterial genome termed that allows for chemical- or light-inducible control of the Short Regularly Spaced Repeats (SRSRs).119,120 More than a enzyme may also aid further implementation of CRISPR to decade after the discovery of these repeats, proteins associated control gene expression.135 with the SRSRs were uncovered, and the observed system was One of the major concerns with any genome editing technology labelled as Clustered Regularly Interspaced Short Palindromic is off-target activity, which could both confound scientific results Repeats (CRISPR).121 Eventually, the origins of the CRISPR and drive toxicities in therapeutic applications. Investigations spacers were traced to bacteriophage and extrachromosomal into the specificity of CRISPR have provided mixed results, with DNA,122–124 supporting the hypothesis that CRISPR is a system suggestions that sgRNA size and concentration can affect the of acquired resistance against viruses and that allows probability of off-target events,136–138 and a computational bacteria to ‘‘memorize’’ sequences associated with threats.125,126 model has been developed for optimal sgRNA design based on The naturally occurring CRISPR requires two RNA molecules: these results.139 Another strategy to improve CRISPR-based a mature crRNA that is complementary to the target sequence, nuclease specificity does not use the native Cas9. Instead, and a tracrRNA that activates Cas9 cleavage of DNA.127–129 The the catalytically inactive dCas9 is fused to the Fok1 nuclease, only requirement for these RNA molecules is that the target which requires the dimeric binding of two sgRNAs to trigger sequence for the crRNA be adjacent to the three base-pair NGG cleavage.140,141 This dimeric binding requirement thus improves sequence, called the PAM sequence. Cas9-mediated cleavage of the specificity of the overall system. a sequence triggers repair mechanisms that can effectively The performance of dCas9–Fok1 is a prime example of how knock out expression of a gene. With the ability to easily target thenativeCRISPRsystemcanbeimproved to suit our engineering genes for knockout, Cas9 contains many appealing traits as a purposes. As synthetic biologists continue to modify and experi- genome editing tool, but further engineering of the enzyme has ment with different applications of Cas9, it is entirely possible been undertaken to make it more widely used. that the current iterations of CRISPR for genome control will not be the forms used in the future. A new CRISPR enzyme Developing CRISPR into an easy-to-use tool Cpf1 has been uncovered with tremendous promise due to its One of the first steps to making an accessible Cas9-based smaller size and potential for more accurate genome editing.142 system was to reduce the number of components required for Furthermore, our understanding of how to design technologies its use. In place of separated tracrRNA and crRNAs, these two around Cas9 is evolving as we gain further insight into the effect strands were combined into one synthetic single guide RNA of sgRNA length. While shorter sgRNAs cannot mediate DNA (sgRNA).129 Due to the simple PAM-adjacent requirement for cleavage, they are able to mediate Cas9–VPR activation of gene a target sequence, the identification and verification of a sgRNA expression143,144 (Fig. 3G). And yet Cas9–VPR still contains the targeted towards a particular gene is easy, especially when catalytic nuclease activity of the original Cas9 enzyme and can compared to zinc finger nucleases and TALENs. This chimeric be applied as a nuclease when targeted towards a sequence with sgRNA design turned a compelling study of bacterial adaptive larger sgRNAs. These results demonstrate that one enzyme can immunity into an easy-to-use tool for genome modification, be programmed towards different purposes based on the size of making CRISPR currently one of the most promising tools of the sgRNA and can even carry out orthogonal knockout and synthetic biology and setting a precedent for further modifications activation of genes in the same cell.143 Whether there are other of the system. strategies to manipulate Cas9 behavior remains to be seen. Zinc fingers and TALEs took on new functions through the CRISPR has been more widely adopted than other genome addition of different effector domains that could trigger nuclease editing technologies because of its efficiency and ease-of-use. activity, gene activation, or gene repression. To turn Cas9 into This is a case where synthetic biology strategies have played an an enzyme of similar potential, a catalytically inactive dCas9 was important role in simplifying the CRISPR workflow, and thus generated.19 The dCas9 enzyme can still be guided towards a helped democratize a technology to make cellular engineering a target sequence using a sgRNA, and while in this catalytically dead widely used tool. The ultimate contribution from CRISPR may form, the dCas9–sgRNA complex can interfere with transcription not be one specific tool, but instead a toolbox that provides and repress gene expression. Similar to zinc fingers and TALEs, scientists with a wide range of options that are appropriate for dCas9 can regulate gene expression by connecting the enzyme different systems and questions. Already, the methodologies to a transcriptional activator (such as VP64) or repressor domain underlined by CRISPR have helped in the rapid generation of

This journal is © The Royal Society of Chemistry 2016 Integr. Biol., 2016, 8, 504--517 | 511 Integrative Biology Review Article complex animal disease models145–149 and is being further with the CD28 domain, this continuous proliferation was only explored for use in gene therapies.150,151 As the arsenal of observed with certain scFvs, though the exact rules describing genome editing tools expands, the scope of scientific questions this effect are still not understood.152 and engineering challenges that can be addressed will also expand. Indeed, there are a number of variables that can affect therapeutic outcomes, including receptor expression level and transduction method. These effects may have predictable con- Broader lessons sequences, but there will likely be responses that require further investigation and characterization. CAR-based therapy Every engineering field strives to produce technologies that reflects a need to understand not just how engineered parts can address real world challenges. This goal requires careful

affect the cell, but also how engineered cells can affect the body. Downloaded from https://academic.oup.com/ib/article-abstract/8/4/504/5163774 by Columbia University user on 27 March 2020 understanding of the rules governing the materials used, a task Complexity does not only originate from the cell, but also from that can be particularly challenging when trying to develop its surroundings, especially when the environment contains the tools based on cellular substrates with ill-defined properties. intersection of physiological systems that affect toxicity, immu- Synthetic biology represents a new frontier of technology, and nogenicity, and functionality. These considerations have long understanding its ability to solve problems in the world is an been important considerations in medicine, and as synthetic ongoing undertaking. The work described in this review high- biology and adoptive immunotherapy seek greater relevance, lights the potential of building novel systems with biological these fields will need to learn how to take the whole body into components. Their success can provide insight into strategies account as a design constraint. that will make synthetic biology more widely applicable. Computational models can also play a powerful role in characterizing the interactions that affect these synthetic systems, Designing better biological tools as evidenced in construction of increasingly robust oscillators. The To design structural buildings and transportation that fulfill iterations of synthetic oscillators relied heavily on theoretical varying requirements, engineers have relied on a careful under- models to determine how tuning different parameters would affect standing of the world they are constructing in. Constructing experimental performance. For example, the repressilator model within a cell thus poses a distinct challenge because of the illustrated the roles of protein synthesis and protein decay rates, many components and processes that are not fully understood. which was instituted experimentally with promoters to vary These unknown factors can wreak havoc on otherwise carefully synthesis rate and a carboxy-terminal tag to control protein planned systems. decay rate.7 Models also helped inform the development of The most direct approach to address this challenge is to stable oscillations in the activator/inhibitor relaxation oscillator92 extensively characterize the known components and interactions and the relationship between glycolytic flux and oscillations in the of the engineered system. For example, varied results between metabolator.99 Beyond just informing parameter constraints, these clinical trials with CARs that use different co-stimulatory domains models also provide information on network structures that have emphasized that to fully take advantage of this system’s improve circuit behavior,155 such as the importance of the delayed modularity, we must understand the parts used. CARs with CD28 negative feedback loop and positive feedback loop towards chains have exhibited faster action and shorter persistence when robustness and tunability in the tunable relaxation oscillator.93 compared to 4-1BB CARs, affecting the onset of cytokine release Computational models have played a role in developing CRISPR syndrome and B cell aplasia in patients.60–62 Further in vitro and into a more powerful tool as well, allowing scientists to design in vivo characterization of CARs containing these domains have sgRNA to more intelligently target endogenous genes.139 These revealed that—dependent on scFv and expression level—CD28 can computational tools are widely available and enable the design mediate constitutive proliferation of T cells prior to target binding, of guide RNAs of optimal length and specificity to avoid a result that may have implications for its use in therapy.152 off-target effects. Another comparison of CD28 and 4-1BB CARs have suggested Distilling biological results into mathematical terminology that CD28 CARs may provide better tumor control at low T cell can often pose a challenge due to the wide range of potential doses compared to 4-1BB.153 This study also tested the performance parameters, but their use underscores a deeper understanding of a newer design that—instead of linking the co-stimulatory of the system being designed. As the field seeks to design domain directly to the receptor—co-expresses 4-1BBL (the robust systems for wider applications, computational models ligand that binds to 4-1BB) with the first generation receptor. will continue to play a vital role in improving and characterizing This receptor/4-1BBL co-expression model exhibited the greatest engineered tools. control of tumors at low T cell dosages, expanding the space of CAR design. Assessing broader world views of synthetic biology Further characterization of the scFv of the CAR can provide While synthetic biology has been able to reduce the cost of drug additional insights into the development of safe and effective production, some global challenges remain to be addressed for therapies. Choosing a lower affinity CAR has the potential to this approach to be viable towards new technologies. The increase T cell selectivity of the tumors,154 implicating the scFv projects in this review also illustrate some of the global affinity as a possible parameter to tune the safety of the therapy. challenges that synthetic biology must address to establish As noted in the study of constitutive proliferation associated itself as a true player in developing technologies. One of the

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